Modification of EDC method for increased labeling efficiency and characterization of low-content protein in gum acacia using asymmetrical flow field-flow fractionation coupled with multiple detectors.

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is widely used as a crosslinker for fluorescence labeling of protein in the fields of biochemistry and food analysis. Many natural polysaccharides often contain some proteins or peptides that are very low in content but play a vital role in their biological function as well as technical applications. Determination of these low-content proteinaceous matters requires a highly sensitive and selective method. In this study, a methodological approach for investigations of the presence of proteinaceous material over the molar mass distribution (MD) of polysaccharides was developed using gum acacia (GA) as a model polysaccharide. EDC fluorescence-labeling method was modified by changing the pH (7, 9, and 11) of the solution for the analysis of low-content protein in food materials. Fluorescence spectroscopy and asymmetrical flow field-flow fractionation (AF4) were employed for characterizing the labeling efficiency and physiochemical properties of unlabeled and fluorescence-labeled GA. AF4 provided molar mass (M) and the radius of gyration (rG) of arabinogalactan (AG) and arabinogalactan protein complex (AGP) and determined the presence of proteinaceous matter over the MD. The labeling efficiencies of GA at pH 7, 9, and 11 determined by fluorescence spectroscopy were 56.5, 68.4, and 72.0%, respectively, with an increment of 15.5% when pH was increased from 7 to 11. The modified EDC fluorescence-labeling method allows highly sensitive and selective analysis of low-content proteinaceous matters and their distribution in natural polysaccharides.

Northern Blot Detection of Tiny RNAs.

Successful detection of very small RNAs (tiny RNAs, ~8-15 nt in length) by northern blotting depends on tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with 5'-digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 5'-32P-end-label. Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate size (~50-400 nt).

Facilitating GL13K Peptide Grafting on Polyetheretherketone via 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide: Surface Properties and Antibacterial Activity.

In the present work, the antimicrobial peptide (AMP) of GL13K was successfully coated onto a polyetheretherketone (PEEK) substrate to investigate its antibacterial activities against Staphylococcus aureus (S. aureus) bacteria. To improve the coating efficiency, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was mixed with a GL13K solution and coated on the PEEK surface for comparison. Both energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS) data confirmed 30% greater peptide coating on PEEK/GL13K-EDC than PEEK without EDC treatment. The GL13K graft levels are depicted in the micrograms per square centimeter range. The PEEK/GL13K-EDC sample showed a smoother and lower roughness (Rq of 0.530 µm) than the PEEK/GL13K (0.634 µm) and PEEK (0.697 µm) samples. The surface of the PEEK/GL13K-EDC was more hydrophilic (with a water contact angle of 24°) than the PEEK/GL13K (40°) and pure PEEK (89°) samples. The pure PEEK disc did not exhibit any inhibition zone against S. aureus. After peptide coating, the samples demonstrated significant zones of inhibition: 28 mm and 25 mm for the PEEK/GL13K-EDC and PEEK/GL13K samples, respectively. The bacteria-challenged PEEK sample showed numerous bacteria clusters, whereas PEEK/GL13K contained a little bacteria and PEEK/GL13K-EDC had no bacterial attachment. The results confirm that the GL13K peptide coating was able to induce antibacterial and biofilm-inhibitory effects. To the best of our knowledge, this is the first report of successful GL13K peptide grafting on a PEEK substrate via EDC coupling. The present work illustrates a facile and promising coating technique for a polymeric surface to provide bactericidal activity and biofilm resistance to medical implantable devices.

Tyrosine-EDC Conjugation, an Undesirable Side Effect of the EDC-Catalyzed Carboxyl Labeling Approach.

Carbodiimide-catalyzed carboxyl and amine conjugation (amidation) has been widely used to protect carboxyl groups. N-(3-(Dimethylamino)propyl)-N'-ethylcarbodiimide (EDC) is the most common carbodiimide reagent in protein chemistry due to its high catalytic efficiency in aqueous media. The reaction has also been applied in different proteomic studies including protein terminomics, glycosylation, and interaction. Herein, we report that the EDC-catalyzed amidation could cause a +155 Da side modification on the tyrosine residue and severely hamper the identification of Tyr-containing peptides. We revealed the extremely low identification rate of Tyr-containing peptides in different published studies employing the EDC-catalyzed amidation. We discovered a +155 Da side modification occurring specifically on Tyr and decoded it as the addition of EDC. Consideration of the side modification in a database search enabled the identification of 13 times more Tyr-containing peptides. Furthermore, we successfully developed an efficient method to remove the side modification. Our results also imply that chemical reactions in proteomic studies should be carefully evaluated prior to their wide applications. Data are available via ProteomeXchange with identifier PXD020042.

Optimization of O-GIG for O-Glycopeptide Characterization with Sialic Acid Linkage Determination.

O-Glycoprotein analysis has been historically challenging due, in part, to a dearth of available enzymes active in the release of O-glycans. Moreover, chemical releasing methods, such as ß-elimination/Michael addition, are not specific to O-glycan release and can also eliminate phosphoryl substitutions. Both of these events leave behind deaminated serine and threonine and thus can lead to ambiguous structural conclusions. Recently, the O-protease OpeRATOR, derived from intestinal bacteria and expressed in Escherichia coli, has become commercially available. The digestion of O-glycoprotein yields O-glycopeptides cleaved at the N-terminal end of serine and threonine, with O-glycan remaining intact. The enzyme has a broad substrate specificity and includes mammalian cores 1-8. However, OpeRATOR is not fully active toward sialylated glycoproteins, and it has been suggested that this acidic residue be removed prior to digestion, thus sacrificing structural information. In this study, we investigated the performance of OpeRATOR under a range of conditions, including buffer selection, varying pH, sialic acid modification, and digestion temperature, in order to optimize the enzymatic activity, with a special emphasis on sialylated glycosites. Conditions derived in this work facilitate the OpeRATOR digestion of fully sialylated O-glycopeptides that are mass tagged to identify the sialyl linkage, thus facilitating the analysis of these charged O-glycopeptides, which are often important in biological processes.

Synergistic improvement in the performance of insect odorant receptor based biosensors in the presence of Orco.

Insect odorant receptors (ORs) are believed to be a complex of an odorant binding subunit, OrX, and an ion channel forming subunit, Orco. In our previous study, we showed that the OrX subunit on its own in liposomes could detect volatile organic compounds (VOCs) ultrasensitively using Electrochemical Impedance Spectroscopy (EIS). In this study, we investigated the effect of the presence of Orco on the response of the OrX subunit to detect the VOCs. The OrXs - Or10a, Or22a, Or35a and Or71a, together with Orco, were recombinantly expressed, purified and integrated into liposomes. These OrX/Orco liposomes were covalently attached to a gold surface modified with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (NHS/EDC)-activated self-assembled monolayers (SAMs) of 6-mercaptohexanoic acid (MHA). It was demonstrated that the OrX/Orco liposomes could sensitively and selectively detect their ligands by monitoring a change in frequency and impedance signal upon binding with both Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and EIS. Using EIS, three OrXs (Or10a, Or22a and Or35a) showed a shift in their dose-response curves when Orco was co-integrated, reflecting an increase in ligand sensitivity and a decrease in limit of detection (LOD). Or71a in the presence of Orco did not show any improvement in ligand sensitivity as this is a highly tuned receptor which may be already at the sensitivity limit for EIS. The observed enhancement in sensor performance is believed to be an effect of Orco which is stabilizing the OrX in a more active conformation and amplifying charge transfer to result in a greater reduction in impedance.

Paclitaxel loaded EDC-crosslinked fibroin nanoparticles: a potential approach for colon cancer treatment.

Colon cancer is one of the most life-threatening cancers with high incidence and mortality rates. Current first-line treatments are ineffective and possess many unwanted effects. The off-label use of paclitaxel encapsulated in nanoparticles proves an innovative approach. In this study, we reported novel paclitaxel loaded EDC-crosslinked fibroin nanoparticles (PTX-FNPs) for anticancer purpose. The particles were formulated using desolvation method and the physicochemical properties were controlled favorably, including the particle size (300-500 nm), zeta potential (- 15 to + 30 mV), drug entrapment efficiency (75-100%), crystallinity, drug solubility (1- to 10-fold increase), dissolution profiles, stability (> 24 h in intravenous diluent and > 6 months storage at 4 °C). In in vitro study, all formulations showed no toxicity on the red blood cells, whereas retained the paclitaxel cytotoxicity on MCF-7 breast cancer and Caco-2 colon cancer cells. Interestingly, PTX-FNPs can be uptaken rapidly by the Caco-2 cells, consequently increased paclitaxel potency up to 10-fold compared to the free drug. Graphical abstract.

Optimizing PLG nanoparticle-peptide delivery platforms for transplantation tolerance using an allogeneic skin transplant model.

A robust regimen for inducing allogeneic transplantation tolerance involves pre-emptive recipient treatment with donor splenocytes (SP) rendered apoptotic by 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide(ECDI) treatment. However, such a regimen is limited by availability of donor cells, cost of cell procurement, and regulatory hurdles associated with cell-based therapies. Nanoparticles (NP) delivering donor antigens are a promising alternative for promoting transplantation tolerance. Here, we used a B6.C-H-2bm12(bm12) to C57BL/6(B6) skin transplant model involving a defined major histocompatibility antigen mismatch to investigate design parameters of poly(lactide-co-glycolide) (PLG) NPs delivering peptides containing the donor antigen for optimizing skin allograft survival. We showed that an epitope-containing short peptide (P1) was more effective than a longer peptide (P2) at providing graft protection. Importantly, the NP and P1 complex (NP-ECDI-P1) resulted in a significant expansion of graft-infiltrating Tregs. Interestingly, in comparison to donor ECDI-SP that provided indefinite graft protection, NP-ECDI-P1 targeted different splenic phagocytes and skin allografts in these recipients harbored significantly more graft-infiltrating CD8+IFN-γ+ cells. Collectively, the current study provides initial engineering parameters for a cell-free and biocompatible NP-peptide platform for transplant immunoregulation. Moreover, it also provides guidance to future NP engineering endeavors to recapitulate the effects of donor ECDI-SP as a goal for maximizing tolerance efficacy of NP formulations.

Cytochrome c-poly(acrylic acid) conjugates with improved peroxidase turnover number.

Cytochrome c-poly(acrylic acid) (cyt c-PAA) conjugates with 34-fold enchancement in peroxidase turnover number (kcat) are reported. Cyt c-PAA conjugates were prepared by carbodiimide coupling. PAA with molecular weight (Mw) ranging from 1.8k to 250k g mol-1 were employed, and the effect of PAA Mw on peroxiodase kinetics was assessed. The kcat value increased with increased Mw of PAA, ranging from 0.077(±0.002) s-1 in the absence of PAA to 2.66(±0.08) s-1 for the conjugate of cyt c with 250k PAA. Enzymatic activity studies over pH 6-8 indicated improved activity for cyt c-PAA conjugates at neutral or slightly alkaline pH. Examination of the cyt c heme spectroscopy in the presence of H2O2 revealed that formation of compound III, a reactive intermediate that leads to enzyme inactivation, was supressed in cyt c-PAA conjugates. Thus, we suggest the kcat enhancement can be attributed to acidification of the pH microenvironment and inhibition of the formation of a reactive intermediate that deactivates cyt c during the catalytic cycle.

Silk fibroin coating through EDC/NHS crosslink is an effective method to promote graft remodeling of a polyethylene terephthalate artificial ligament.

Anterior cruciate ligament reconstruction using polyethylene terephthalate artificial ligaments is one of the research hotspots in sports medicine but it is still challenging to achieve biological healing. The purpose of this study was to modify polyethylene terephthalate ligament with silk fibroin through ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) crosslink and to investigate the performance of graft remodeling in vitro and in vivo. After silk fibroin coating, changes in the surface properties of ligament were characterized by scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy and water contact angle measurements. The compatibility of polyethylene terephthalate ligament with silk fibroin coating was investigated in vitro. The results showed the silk fibroin coating significantly improved adhesion, proliferation and extracellular matrix secretion of fibroblast cells. Moreover, a rabbit anterior cruciate ligament reconstruction model was established to evaluate the effect of ligament with silk fibroin coating in vivo. The gross observation and histological results showed that the silk fibroin coating significantly inhibited inflammation response and promoted new tissue regeneration with fusiform cells infiltration in and around the graft. Furthermore, the expressions of collagen I protein and mRNA in the silk fibroin-coated polyethylene terephthalate group were much higher than those in the control group according to the immunohistochemical and real-time polymerase chain reaction results. Therefore, silk fibroin coating through EDC/NHS crosslink promotes the biocompatibility and remodeling process of polyethylene terephthalate artificial ligament in vitro and in vivo. It can be considered as a potential solution to the problem of poor remodeling of artificial ligaments after anterior cruciate ligament reconstruction in the clinical applications.

Investigating Possible Enzymatic Degradation on Polymer Shells around Inorganic Nanoparticles.

Inorganic iron oxide nanoparticle cores as model systems for inorganic nanoparticles were coated with shells of amphiphilic polymers, to which organic fluorophores were linked with different conjugation chemistries, including 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry and two types of "click chemistry". The nanoparticle-dye conjugates were exposed to different enzymes/enzyme mixtures in order to investigate potential enzymatic degradation of the fluorophore-modified polymer shell. The release of the dyes and polymer fragments upon enzymatic digestion was quantified by using fluorescence spectroscopy. The data indicate that enzymatic cleavage of the fluorophore-modified organic surface coating around the inorganic nanoparticles in fact depends on the used conjugation chemistry, together with the types of enzymes to which the nanoparticle-dye conjugates are exposed.

Electrospinning in water and in situ crosslinking of hyaluronic acid / cyclodextrin nanofibers: Towards wound dressing with controlled drug release.

Hyaluronic acid (HA) is widely investigated due to its high potential for wound dressing applications. The fabrication of biomimetic HA-based scaffolds by electrospinning is thus extensively studied. However, HA is often dissolved in toxic organic solvents to allow the efficient production of electrospun nanofibers. Indeed, although HA is soluble in water, its ionic nature leading to long-range electrostatic interactions and the presence of counter ions induce a dramatic increase of the viscosity of aqueous HA solutions without insuring enough chain entanglements necessary for a stable and efficient electrospinning. In this study, biocompatible insoluble HA-based nanofibers were fabricated by electrospinning in pure water. To this end, poly(vinyl alcohol) (PVA) was added as a carrier polymer and it was found that the addition of hydroxypropyl-ßcyclodextrin (HPßCD) stabilized the process of electrospinning and led to the efficient formation of uniform nanofibrous scaffolds. An in situ crosslinking process of the scaffolds is also proposed, insuring a whole fabrication process without any toxicity. Furthermore, the beneficial presence of HPßCD in the HA-based scaffolds paves the way for wound dressing applications with controlled drug encapsulation-release properties. As a proof of concept, naproxen (NAP), a non-steroidal anti-inflammatory drug was chosen as a model drug. NAP was impregnated into the scaffolds either in aqueous solution or under supercritical CO2. The resulting functional scaffolds showed a regular drug release profile along several days without losing the fibrous structure. This study proposes a simple approach to form stable HA-based nanofibrous scaffolds embedding HPßCD using water as the only solvent, enabling the development of safe functional wound dressings.

EGCG and enzymatic cross-linking combined treatments for improving elastin stability and reducing calcification in bioprosthetic heart valves.

The failures of glutaraldehyde (GLUT) cross-linked bioprosthetic heart valves (BHVs) are mainly due to degeneration and calcification. In this study, we developed a new preparation strategy for BHVs named as "HPA/EDC/EGCG" that utilized 3,4-hydroxyphenylpropionic acid (HPA)-conjugated pericardium, epigallocatechin gallate (EGCG), and horseradish peroxidase (HRP)/hydrogen peroxide (H2 O2 ) enzymatic cross-linking. HPA-pericardium conjugation was done by carbodiimide coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then HPA-conjugated pericardium was cross-linked by HRP/H2 O2 enzyme-catalyzed oxidation. The feeding ratios of HPA and EGCG were optimized. The consumption of amino groups, collagenase and elastase degradation in vitro, biomechanics, extracellular matrix stability, and calcification of HPA-/EDC-/EGCG-treated pericardiums were characterized. We demonstrated that HPA-/EDC-/EGCG-treated pericardiums had better elastin stabilization and less calcification. EGCG and enzymatic cross-linking treated pericardiums showed improved mechanical properties. This new EGCG and enzymatic cross-linking strategy would be a promising method to make BHVs with better elastin stability and anti-calcification property. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1551-1559, 2019.

Real-time circulating tumor cells detection via highly sensitive needle-like cytosensor-demonstrated by a blood flow simulation.

The concept of rapid detection of circulating tumor cells (CTCs) has always been the focal point of modern and future medicine. However, the dispersity and rarity of CTCs in the bloodstream makes it hard to detect metastasis. Herein, our newly designed needle-like cytosensor demonstrates that the capture and analysis of CTCs are a much less laborious process and have more potential than ever. Our aim is to detect and capture CTCs directly in the bloodstream without altering the genetic information; further benefit of current cytosensor is allows for the whole circulation of blood to run through the cytosensor, giving a much better sensitivity and chance of detecting CTCs. Our functionalized needle-like cytosensor has been modified with 3-aminopropyltriethoxysilane, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide and conjugated streptavidin to allow the binding of the biotinylated-antibody of epithelial cell adhesion molecules, which captures targeted colon cancer CTC. The capability of our needle-like cytosensor to detect CTCs spanned from 102 to 106 cells/mL. Beyond this, the needle-like cytosensor avoids the distortion of the cell information. In addition, we constructed a blood flow simulation that mimics human circulating system about 10 mL/min speed; by using cyclic voltammetry we could detect significant signals from captured cancer CTCs more than 21 cells/mL without delay; the fluorescence dye detection was further performed for data confirmation. The future of biosensors begins with this, by providing early monitoring quality care in cancer therapy.

A fluorescent sensor based on thioglycolic acid capped cadmium sulfide quantum dots for the determination of dopamine.

A fluorescent sensor based on thioglycolic acid-capped cadmium sulfide quantum dots (TGA-CdS QDs) has been designed for the sensitive and selective detection of dopamine (DA). In the presence of dopamine (DA), the addition of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) activates the reaction between the carboxylic group of the TGA and the amino group of dopamine to form an amide bond, quenching the fluorescence of the QDs. The fluorescence intensity of TGA-CdS QDs can be used to sense the presence of dopamine with a limit of detection of 0.68µM and a working linear range of 1.0-17.5µM. This sensor system shows great potential application for dopamine detection in dopamine drug samples and for future easy-to-make analytical devices.

Enhancement in Biological Activity of L-Asparginase by its Conjugation on Silica Nanoparticles.

BACKGROUND: L-asparaginase is a drug of choice in the treatment of Hodgkin's lymphoma and acute lymphoblastic leukemia. Production of its bioconjugates can increase its half-life, stability and decrease its immunogenicity. OBJECTIVE: The aim of the present study was to immobilize this drug on silica nanoparticles by two different cross-linking agents. METHOD: The drug was conjugated to nanoparticles by two cross-linking agents; 1-ethyl-3-(3- dimethylaminopropyl) carboiimide HCl (EDC) or glutaraldehyde. The effect of the drug to the nanoparticles ratio, the amount of cross-linking agents and the time of conjugation were optimized according to the zeta potential, size particle and the enzyme immobilization efficiency. Conjugation of L-asparaginase to nanoparticles was confirmed by FT-IR and TEM. The activity, kinetic profiles, stability against pH changes, thermal and storage stability of the native and immobilized drug were compared. RESULTS: The results showed significant increase in pH range of the stability and decrease in the km value of the drug after immobilization; indicating an increase in the enzyme tendency for the substrate. The Time of stability of the drug increased after immobilization in plasma and phosphate buffer saline which can increase its half-life of circulation. CONCLUSION: The activity and stability of immobilized drug by EDC were better than glutaraldehyde.

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase.

Using glucose oxidase (GOx) and α-Zr(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme-solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx(n), where n is the net charge on the enzyme after the modification and ranged from -62 to +95 electrostatic units in the system. The binding of GOx(n) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx(n) where n ≥ -20 showed binding affinities exceeding (2.1 ± 0.6) × 106 M-1, resulting from the charge modification of the enzyme. A plot of GOx(n) charge vs Gibbs free energy of binding (ΔG) for n = +20 to n = +65 indicated an overall increase in favorable interaction between GOx(n) and α-ZrP nanosheets. However, ΔG is less dependent on the net charge for n > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme-nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis.

Fundamental insight into the effect of carbodiimide crosslinking on cellular recognition of collagen-based scaffolds.

Research on the development of collagen constructs is extremely important in the field of tissue engineering. Collagen scaffolds for numerous tissue engineering applications are frequently crosslinked with 1-ethyl-3-(3-dimethylaminopropyl-carbodiimide hydrochloride (EDC) in the presence of N-hydroxy-succinimide (NHS). Despite producing scaffolds with good biocompatibility and low cellular toxicity the influence of EDC/NHS crosslinking on the cell interactive properties of collagen has been overlooked. Here we have extensively studied the interaction of model cell lines with collagen I-based materials after crosslinking with different ratios of EDC in relation to the number of carboxylic acid residues on collagen. Divalent cation-dependent cell adhesion, via integrins α1ß1, α2ß1, α10ß1 and α11ß1, were sensitive to EDC crosslinking. With increasing EDC concentration, this was replaced with cation-independent adhesion. These results were replicated using purified recombinant I domains derived from integrin α1 and α2 subunits. Integrin α2ß1-mediated cell spreading, apoptosis and proliferation were all heavily influenced by EDC crosslinking of collagen. Data from this rigorous study provides an exciting new insight that EDC/NHS crosslinking is utilising the same carboxylic side chain chemistry that is vital for native-like integrin-mediated cell interactions. Due to the ubiquitous usage of EDC/NHS crosslinked collagen for biomaterials fabrication this data is essential to have a full understanding in order to ensure optimized collagen-based material performance. STATEMENT OF SIGNIFICANCE: Carbodiimide stabilised collagen is employed extensively for the fabrication of biologically active materials. Despite this common usage, the effect of carbodiimide crosslinking on cell-collagen interactions is unclear. Here we have found that carbodiimide crosslinking of collagen inhibits native-like, whilst increasing non-native like, cellular interactions. We propose a mechanistic model in which carbodiimide modifies the carboxylic acid groups on collagen that are essential for cell binding. As such we feel that this research provides a crucial, long awaited, insight into the bioactivity of carbodiimide crosslinked collagen. Through the ubiquitous use of collagen as a cellular substrate we feel that this is fundamental to a wide range of research activity with high impact across a broad range of disciplines.

A Biomimetic Silk Fibroin/Sodium Alginate Composite Scaffold for Soft Tissue Engineering.

A cytocompatible porous scaffold mimicking the properties of extracellular matrices (ECMs) has great potential in promoting cellular attachment and proliferation for tissue regeneration. A biomimetic scaffold was prepared using silk fibroin (SF)/sodium alginate (SA) in which regular and uniform pore morphology can be formed through a facile freeze-dried method. The scanning electron microscopy (SEM) studies showed the presence of interconnected pores, mostly spread over the entire scaffold with pore diameter around 54~532 µm and porosity 66~94%. With significantly better water stability and high swelling ratios, the blend scaffolds crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) provided sufficient time for the formation of neo-tissue and ECMs during tissue regeneration. Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) results confirmed random coil structure and silk I conformation were maintained in the blend scaffolds. What's more, FI-TR spectra demonstrated crosslinking reactions occurred actually among EDC, SF and SA macromolecules, which kept integrity of the scaffolds under physiological environment. The suitable pore structure and improved equilibrium swelling capacity of this scaffold could imitate biochemical cues of natural skin ECMs for guiding spatial organization and proliferation of cells in vitro, indicating its potential candidate material for soft tissue engineering.

A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2.

Silicon-substituted calcium phosphate (Si-CaP) was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP) cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2) was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future.